U.S. patent number 5,639,635 [Application Number 08/333,912] was granted by the patent office on 1997-06-17 for process for bacterial production of polypeptides.
This patent grant is currently assigned to Genentech, Inc.. Invention is credited to John C. Joly, James R. Swartz.
United States Patent |
5,639,635 |
Joly , et al. |
June 17, 1997 |
Process for bacterial production of polypeptides
Abstract
A process is provided for producing a heterologous polypeptide
in bacteria, which process comprises: (a) culturing bacterial
cells, which cells comprise nucleic acid encoding a DsbA or DsbC
protein, nucleic acid encoding the heterologous polypeptide, a
signal sequence for secretion of both the DsbA or DsbC protein and
the heterologous polypeptide, and an inducible promoter for both
the nucleic acid encoding the DsbA or DsbC protein and the nucleic
acid encoding the heterologous polypeptide, under conditions
whereby expression of the nucleic acid encoding the DsbA or DsbC
protein is induced prior to induction of the expression of the
nucleic acid encoding the heterologous polypeptide, and under
conditions whereby either both the heterologous polypeptide and the
DsbA or DsbC protein are secreted into the periplasm of the
bacteria or the heterologous polypeptide is secreted into the
medium in which the bacterial cells are cultured; and (b)
recovering the heterologous polypeptide from the periplasm or the
culture medium.
Inventors: |
Joly; John C. (San Mateo,
CA), Swartz; James R. (Menlo Park, CA) |
Assignee: |
Genentech, Inc. (South San
Francisco, CA)
|
Family
ID: |
23304776 |
Appl.
No.: |
08/333,912 |
Filed: |
November 3, 1994 |
Current U.S.
Class: |
435/69.1;
536/23.5; 536/23.6; 536/23.7 |
Current CPC
Class: |
C07K
1/1133 (20130101); C07K 14/245 (20130101); C07K
14/65 (20130101); C12N 9/0051 (20130101); C12N
9/90 (20130101); C12N 15/70 (20130101); C12P
21/02 (20130101); C07K 2319/00 (20130101) |
Current International
Class: |
C07K
14/195 (20060101); C07K 1/113 (20060101); C07K
14/245 (20060101); C07K 1/00 (20060101); C07K
14/435 (20060101); C07K 14/65 (20060101); C12N
9/90 (20060101); C12P 21/02 (20060101); C12N
15/70 (20060101); C12N 9/02 (20060101); C12P
021/06 () |
Field of
Search: |
;435/69.7,69.1,252.3,252.33,320.1 ;536/27,23.7,23.5,23.6 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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293 793 A2 |
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Dec 1988 |
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EP |
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510658 |
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Oct 1992 |
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EP |
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509 841 |
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Oct 1992 |
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EP |
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60-38771 |
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Feb 1994 |
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JP |
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WO 93/25676 |
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Dec 1993 |
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WO |
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WO 94/08012 |
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Apr 1994 |
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WO |
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Other References
Chivers et al., "The CXXC Motif: Imperatives for the Formation of
Native Disulfide Bonds in the Cell" The EMBO Journal
15(11):2659-2667 (1996). .
Grauschopf et al., "Why is DsbA Such an Oxidizing Disulfide
Catalyst?" Cell 83:947-955 (1995). .
Bardwell et al., "The Bonds That Tie: Catalyzed Disulfide Bond
Formation" Cell 74:769-771 (1993). .
Bardwell et al., "Identification of a Protein Required for
Disulfide Bond Formation In Vivo" Cell 67:581-589 (1991). .
Bardwell et al., "A pathway for disulfide bond formation in vivo"
Proc. Natl. Acad. Sci 90:1038-1042 (1993). .
Bulleid, Neil J., "Protein Disulfide-Isomerase: Role in
Biosynthesis of Secretory Proteins" Adv. Prot. Chem. 44:125-150
(1993). .
Creighton et al., "Catalysis by Protein-disulphide Isomerase of the
Unfolding and Refolding of Proteins with Disulphide Bonds" J. Mol.
Biol. 142:43-62 (1980). .
Freedman et al., "Role of Protein Disulphide-Isomerase in the
Expression of Native Proteins" Biochem. Soc. Symp. 55:167-192
(1989). .
Kamitani et al., "Identification and characterization of an
Escherichia coli gene required for the formation of correctly
folded alkaline phosphates a periplasmic enzyme" The EMBO Journal
11(1):57-62 (1992). .
Knappik et al., "The Effect of Folding Catalysts on the In Vivo
Folding Process of Different Antibody Fragments Expressed in
Eschericha coli " Bio/Technology 11:77-83 (1993). .
LaMantia et al., "The Essential Function of Yeast Protein Disulfide
Isomerase Does Not Reside in Its Isomerase Activity" Cell
74:899-908 (1993). .
Martin et al., "Crystal structure of the DsbA protein required for
disulphide bond formation in vivo" Nature 365:464-468 (1993). .
Missiakas et al., "The Escherichia coli dsbC (xprA) gene encodes a
periplasmic protein involved in disulfide bond formation" The EMBO
Journal 3(8):2013-2020 (1994). .
Missiakas et al., "Identification and characterization of the
Escherichia coli gene dsbB, whose product is involved in the
formation of disulfide bonds in vivo" Proc. Natl. Acad. sci.
90:7084-7088 (1993). .
Noiva et al., "Protein Disulfide Isomerase" Journal of biological
chemistry 267(6):3553-3556 (1992). .
Shevchik et al., "characterization of DsbC, a periplasmic protein
of Erwina chrysanthemi and Escherichia coli with disulfide
isomerase activity" The EMBO Journal 13(8):2007-2012 (1994). .
Wulfing et al., "correctly Folded T-cell Receptor Fragments in the
Periplasm of Escherichia coli" J. Mil. Biol. 242:655-669 (1994).
.
Wulfing et al., "Protein folding in the periplasm of Escherichia
coli" Molecular Microbiology 12 (5):685-692 (1994). .
Wunderlich et al., "In Vivo Control of Redox Potential During
Protein Folding Catalysed by Bacterial Protein Disulfide-isomerase
(DsbA)" Journal of Biological Chemistry 268(33):24547-24550 (1993).
.
Wunderlich et al., "Redox properties of protein disulfide isomerase
(DsbA) from Escherichia coli" Protein Science 2:717-726 (1993).
.
Zapun et al., "The Reactive and Detabilizing Disulfide Bond of
DsbA, a Protein Required for Protein Disulfide Bond Formation in
Vivo" Biochemistry 32:5083-5092 (1993)..
|
Primary Examiner: Hendricks; Keith D.
Attorney, Agent or Firm: Hasak; Janet E.
Claims
What is claimed is:
1. A process for producing a heterologous polypeptide in bacteria
comprising:
(a) culturing bacterial cells, wherein said bacterial cells
comprise nucleic acid encoding a DsbA or DsbC protein, nucleic acid
encoding the heterologous polypeptide, a signal sequence for
secretion of both the DsbA or DsbC protein and the heterologous
polypeptide, and separate inducible promoters for both the nucleic
acid encoding the DsbA or DsbC protein and the nucleic acid
encoding the heterologous polypeptide, under conditions whereby
expression of the nucleic acid encoding the DsbA or DsbC protein is
induced prior to induction of the expression of the nucleic acid
encoding the heterologous polypeptide, and under conditions whereby
either both the heterologous polypeptide and the DsbA or DsbC
protein are secreted into the periplasm of the bacteria or the
heterologous polypeptide is secreted into the medium in which the
bacterial cells are cultured; and
(b) recovering the heterologous polypeptide from the periplasm or
the culture medium.
2. The process of claim 1 wherein the heterologous polypeptide is a
mammalian polypeptide.
3. The process of claim 2 wherein the mammalian polypeptide is a
human polypeptide.
4. The process of claim 2 wherein the heterologous polypeptide is
insulin-like growth factor.
5. The process of claim 1 wherein the bacterial cells are
eubacterial cells.
6. The process of claim 5 wherein the eubacterial cells are
Enterobacteriaceae cells.
7. The process of claim 6 wherein the eubacterial cells are E. coli
cells.
8. The process of claim 1 wherein nucleic acid encoding DsbA is
expressed.
9. The process of claim 1 wherein the polypeptide is recovered from
the periplasm of the bacterial cells.
10. The process of claim 1 wherein after the polypeptide is
recovered, the DsbA or DsbC protein is separated from the
polypeptide.
11. The process of claim 1 wherein the bacterial cells are
transformed with one or two expression vectors containing the
nucleic acid encoding the DsbA or DsbC protein and the nucleic acid
encoding the heterologous polypeptide.
12. The process of claim 11 wherein the bacterial cells are
transformed with two vectors respectively containing the nucleic
acid encoding the DsbA or DsbC protein and the nucleic acid
encoding the heterologous polypeptide.
13. The process of claim 11 wherein the nucleic acid encoding the
DsbA or DsbC protein and the nucleic acid encoding the heterologous
polypeptide are contained on one vector with which the bacterial
cells are transformed.
14. The process of claim 1 wherein the induction of expression of
the nucleic acid encoding DsbA or DsbC is carried out by adding an
inducer to the culture medium.
15. The process of claim 14 wherein the inducer is IPTG, lactose,
or L-arabinose.
16. The process of claim 1 wherein the culturing is performed in
the absence of glutathione.
17. The process of claim 1 wherein yield of total polypeptide is
increased as a result of the process, and yield of soluble
polypeptide is not changed or is decreased.
18. The process of claim 1 wherein the culturing is carried out
without enhanced levels of expression of nucleic acid encoding a
heat-shock transcription factor over endogenous levels of
expression of said nucleic acid.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a process for producing and secreting
polypeptides in bacteria by overproduction and secretion of a DsbA
or DsbC protein.
2. Description of Related Disclosures
Much research has been conducted in the field of protein folding
since the seminal publication of Anfinsen et al., Proc. Natl. Acad.
Sci. USA, 47: 1309-1314 (1961) showing that, in vitro, reduced and
denatured ribonuclease could refold into the active enzyme with the
formation of suitable disulfide bonds. Later, a catalyst
responsible for oxidative folding in eukaryotes was discovered,
called protein disulfide isomerase (PDI).
Two types of proteins that assist in protein folding have been
described: non-catalytic molecular chaperones that presumably
prevent improper interactions leading to aggregation and events
other than proper folding, and catalysts for two steps in protein
folding, cis-trans prolyl isomerization and disulfide bond
formation. While clear evidence for an in vivo requirement of
prolyl isomerase activity is still lacking, the relatively recent
isolation of mutants that are severely defective in disulfide bond
formation has confirmed that this latter folding step is catalyzed
in vivo.
PDI has been implicated in the catalysis of disulfide bond
formation and rearrangement through in vitro dam. Creighton et al.,
J. Mol. Biol., 142: 43-62 (1980); Freedman et al., Biochem. Soc.
Symp., 55: 167-192 (1989). See also Bardwell and Beckwith, Cell,
74: 769-771 (1993). In addition, yeast mutants in PDI that fail to
form disulfide bonds in carboxypeptidase Y have been identified,
indicating the importance of PDI in disulfide bond formation in
vivo. LaMantia and Lennarz, Cell, 74: 899-908 (1993). In
prokaryotes, mutations in a specific gene identified in the
periplasm of E. coli by three independent investigating groups show
a dramatic and pleiotropic decrease in the rate of disulfide bond
formation in secreted proteins. The protein encoded by this gene
was variously designated as DsbA (disulfide bond) by Bardwell et
al., Cell, 67: 581-589 (1991) and Missiakas et al., Proc. Natl.
Acad. Sci. USA, 90: 7084-7088 (1993), and as PpfA (periplasmic
phosphatase/protein formation) by Kamitani et al., EMBO J., 11:
57-62 (1992). It is hereinafter referred to by the Dsb designation.
The secreted proteins affected by this decrease in disulfide bond
formation range from proteins endogenous to E. coli such as
alkaline phosphatase and OmpA to the recombinant mammalian
proteins, urokinase and tissue plasminogen activator. It is now
clear that the formation of disulfide bonds is a catalyzed process
in both eukaryotes and prokaryotes. For a review on PDI and DsbA,
see Bardwell and Beckwith, Cell, supra (1993).
The DsbA protein has a highly reactive disulfide bond, similar to
disulfide isomerase. Noiva and Lennarz, J. Biol. Chem., 267:
3553-3556 (1992); Bulleld, Adv. Prot., 44: 125-150 (1993). Its
crystal structure has recently been solved (Martin et al., Nature,
365: 464-468 [1993]) and its redox potential has been determined.
Wunderlich and Glockshuber, Prot. Sci., 2: 717-726 (1993). It is a
significantly stronger oxidant than the cytoplasmic thioredoxin and
it more closely resembles the eukaryotic disulfide isomerases. It
was found that unfolding DsbA stabilizes the reactive disulfide
bond by about 18.9 kJmol.sup.-1. Zapun et al., Biochemistry, 32:
5083-5092 (1993). This result is interpreted to indicate that the
disulfide bond destabilizes the folded form of DsbA, thereby
conferring its high energy content.
The gene of a second protein involved in disulfide bond formation,
called dsbB, was cloned by two independent groups. Bardwell et al.,
Proc. Natl. Atari. Sci. USA, 90: 1038-1042 (1993) and Missiakas et
al., 1993, supra. DsbB is an integral membrane protein spanning the
inner membrane. It appears to be involved in the re-oxidation of
DsbA and thus may form pan of a chain that links an electron
transfer step to the formation of disulfide bonds in the periplasm.
Bardwell et al., supra (1993). A third gene for disulfide bond
formation, dsbC, was recently identified. Missiakas et al., EMBO
J., 13: 2013-2020 (1994); Shevchik et al., EMBO J., 13: 2007-2012
(1994). It encodes a 26-kDa periplasmic protein that can
functionally substitute for DsbA.
It has been shown that DsbA is required for the rapid formation of
disulfide bonds of periplasmic E. coli proteins in vivo and in
vitro. The same was found for the production of recombinant
eukaryotic proteins in the periplasm of E. coli, e.g., different
serine proteases (Bardwell et al., supra [1993]), antibody
fragments (Knappik et al., Bio/Technology, 11: 77-83 [1993]), and
fragments of a T-cell receptor. The fraction of the recombinant
molecules that becomes correctly folded can be smaller than in the
case of natural periplasmic proteins, and is very sequence
dependent. Overproduction of DsbA did not help to increase the
proportion of correctly folded periplasmic antibody fragments
(Knappik et al., supra), indicating that other steps limit their
folding in the periplasm. Furthermore, folding of the
.alpha.-amylase/trypsin inhibitor from Ragi in the periplasm was
not improved when the dsbA gene was co-expressed and DsbA protein
co-secreted without reduced glutathione present in the growth
medium, but an increase in correctly folded secreted inhibitor was
observed by co-expression of dsbA in conjunction with the addition
of reduced glutathione to the medium. Wunderlich and Glockshuber,
J. Biol. Chem., 268: 24547-24550 (1993). Further, Wunderlich and
Glockshuber show no increase in total accumulation of the secreted
.alpha.-amylase/trypsin inhibitor protein. Wulfing and Pluckthun,
Molecular Microbiology, 12: 685-692 (1994) produced functional
fragments of a T-cell receptor (TCR) in the periplasm of E. coli by
modest overproduction of DsbA and the E. coli heat-shock proteins
at low temperature. The latter was achieved by over-expression of
rpoH, which codes for the heat-shock sigma factor, sigma.sup.32.
This increased the folding yield of the TCR fragments in the
periplasm by about two orders of magnitude. It is not known what
the yield is in the absence of rpoH overproduction. U.S. Pat. Nos.
5,270,181 and 5,292,646 disclose recombinant production of
heterologous proteins by expression as a fusion protein with a
thioredoxin-like protein (such as the thioredoxin-like domain of
PDI) for high stability and solubility. JP 60-38771 published Feb.
15, 1994 discloses the expression of a human PDI gene linked to
human serum albumin pre-pro sequence and co-expression of this
linked gene and a foreign gene encoding a polypeptide. WO 93/25676
published Dec. 23, 1993 discloses the production of
disulfide-bonded recombinant proteins using a PDI, preferably a
yeast PDI. EP 293,793 published Dec. 7, 1988 discloses a
polypeptide with PDI activity ensuring natural disulfide bridge
arrangement in recombinant proteins. WO 94/08012 published Apr. 14,
1994 discloses increasing secretion of over-expressed gene products
by co-expression of a chaperone protein such as a heat-shock
protein or PDI. EP 509,841 published Oct. 21, 1992 discloses
increased secretion of human serum albumin from yeast cells using a
co-expression system involving PDI and a protein.
There is a continuing need for increasing the total yield of
proteins secreted by prokaryotes.
Therefore, it is an object of this invention to provide a method
for increasing polypeptide yield using an economically viable
method involving physiologically regulating the intracellular
environment for enhanced accumulation of foreign polypeptides in
bacteria.
This object and other objects will become apparent to those skilled
in the art.
SUMMARY OF THE INVENTION
Accordingly, in one aspect the present invention provides a process
for producing a heterologous polypeptide in bacteria
comprising:
(a) culturing bacterial cells, which cells comprise nucleic acid
encoding a DsbA or DsbC protein, nucleic acid encoding the
heterologous polypeptide, a signal sequence for secretion of both
the DsbA or DsbC protein and the heterologous polypeptide, and an
inducible promoter for both the nucleic acid encoding the DsbA or
DsbC protein and the nucleic acid encoding the heterologous
polypeptide, under conditions whereby expression of the nucleic
acid encoding the DsbA or DsbC protein is induced prior to
induction of the expression of the nucleic acid encoding the
heterologous polypeptide, and under conditions whereby either both
the heterologous polypeptide and the DsbA or DsbC protein are
secreted into the periplasm of the bacteria or the heterologous
polypeptide is secreted into the medium in which the bacterial
cells are cultured; and
(b) recovering the heterologous polypeptide from the periplasm or
the culture medium.
The over-expression and secretion of the bacterial protein DsbA or
DsbC results in a significant increase in the amount of
heterologous polypeptide produced in the periplasm of bacteria or
in the culture medium. In the specific example shown below, the
over-expression and secretion of the E. coli protein DsbA resulted
in a large increase in the total yield of human IGF-I deposited in
inclusion bodies in the periplasmic space of E. coli. Furthermore,
this total yield increase is accomplished by culturing in the
absence of any glutathione in the medium and without co-expressing
or over-expressing the E. coli heatshock transcription factor,
RpoH.
While DsbA has been suggested to be involved in the folding of
periplasmic and outer wall proteins of E. coli, it was not known
whether over-expression of the gene encoding this protein would
have any effect, by itself, on secreted human proteins. It was also
not known if over-expression of the dsbA gene would result in
higher levels of DsbA-associated activity. If any effect were seen,
it was expected that higher levels of DsbA activity would promote
the proper folding of IGF-I polypeptide and result in higher levels
of folded, soluble IGF-I monomer. In contrast, a significant
increase was observed in total yield of IGF-I, due to an increase
in the deposition of IGF-I polypeptide into insoluble aggregates,
and the yield of soluble, properly folded protein decreased. Due to
the fact that efficient methods exist for folding these insoluble
aggregates into bioactive polypeptide, this increase in insoluble
polypeptide is a very useful result.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts plasmids employed in the construction of pJJ42, used
to produce IGF-I, namely pBKIGF-2B, pJJ41, and pKMtacII.
FIG. 2 depicts the construction of plasmid pJJ40, used to produce
IGF-I.
FIG. 3 depicts the construction of plasmid pBKIGF2B-A, used to
produce IGF-I.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A. Definitions
As used herein, "dsbA" and "dsbC" refer to genes encoding the
bacterial periplasmic proteins known in the literature as DsbA or
DsbC proteins, respectively. They may be from any bacterial source,
with one example being the dsbA gene from E. coli as described by
Bardwell et al., supra (1991) and Kamitani et al., supra, and the
dsbC gene from E. coli as described by Missiakas et al., 1994,
supra. The dsbA and dsbC genes and their products as used herein do
not include PDI or any other eukaryotic protein that functions as a
protein disulfide isomerase, including those disclosed in U.S. Pat.
Nos. 5,270,181 and 5,292,646; JP 60-38771; WO 93/25676; EP 293,793;
WO 94/08012; and EP 509,841, all supra. DsbA and DsbC differ in
many respects from PDI. For example, DsbA and DsbC retain their
isomerase activity in the absence of redox buffer, whereas PDI
requires a redox buffer for its isomerase activity. Further, DsbA
and DsbC have molecular weights by sequence analysis of 21 kD and
25 kD, respectively, and contain two and four cysteine residues,
respectively, whereas PDI has a molecular weight by sequence
analysis of 57 kD and contains six cysteine residues.
As used herein, "signal sequence" or "signal polypeptide" refers to
a peptide that can be used to secrete the heterologous polypeptide
into the periplasm or medium of the cultured bacteria or to secrete
the DsbA or dsbC protein into the periplasm. The signals for the
heterologous polypeptide may be homologous to the bacteria, or they
may be heterologous, including signals native to the polypeptide
being produced in the bacteria. For the dsbA and dsbC, the signal
sequence is typically that which is endogenous to the bacterial
cells, although it need not be as long as it is effective for its
purpose.
An "over-expressed" gene product is one that is expressed at levels
greater than normal endogenous expression for that gene product. It
can be accomplished, e.g., by introducing a recombinant
construction that directs expression of a gene product in a host
cell, or by altering basal levels of expression of an endogenous
gene product, e.g., by inducing its transcription.
The promoters of this invention are "inducible" promoters, i.e.,
promoters which direct transcription at an increased or decreased
rate upon binding of a transcription factor. "Transcription
factors" as used herein include any factors that can bind to a
regulatory or control region of a promoter and thereby effect
transcription. The synthesis or the promoter binding ability of a
transcription factor within the host cell can be controlled by
exposing the host to an "inducer" or removing an inducer from the
host cell medium. Accordingly, to regulate expression of an
inducible promoter, an inducer is added or removed from the growth
medium of the host cell.
As used herein, the phrase "induce expression" means to increase
the amount of transcription from specific genes by exposure of the
cells containing such genes to an effector or inducer.
An "inducer" is a chemical or physical agent which, when given to a
population of cells, will increase the amount of transcription from
specific genes. These are usually small molecules whose effects are
specific to particular operons or groups of genes, and can include
sugars, phosphate, alcohol, metal ions, hormones, heat, cold, and
the like. For example, isopropylthio-.beta.-galactoside (IPTG) and
lactose are inducers of the tacII promoter, and L-arabinose is a
suitable inducer of the arabinose promoter. The pho gene promoter,
such as phoA and pho5, is inducible by low phosphate concentrations
in the medium.
As used herein, "polypeptide" or "polypeptide of interest" refers
generally to peptides and proteins having more than about ten amino
acids. Preferably, the polypeptides are "exogenous," meaning that
they are "heterologous," i.e., foreign to the host cell being
utilized, such as human protien produced by a CHO cell, or a yeast
polypepide produced by a mammalian cell, or a human polypeptide
produced from a human cell line that is not the native source of
the polypeptide.
Examples of mammalian polypeptides include molecules such as, e.g.,
renin, a growth hormone, including human growth hormone; bovine
growth hormone; growth hormone releasing factor; parathyroid
hormone; thyroid stimulating hormone; lipoproteins;
.alpha.1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;
thrombopoietin; follicle stimulating hormone; calcitonin;
luteinizing hormone; glucagon; clotting factors such as factor
VIIIC, factor IX, tissue factor, and von Willebrands factor;
anti-clotting factors such as Protein C; atrial naturietic factor;
lung surfactant; a plasminogen activator, such as urokinase or
human urine or tissue-type plasminogen activator (t-PA); bombesin;
thrombin; hemopoietic growth factor; tumor necrosis factor-alpha
and -beta; enkephalinase; a serum albumin such as human serum
albumin; mullerjan-inhibiting substance; relaxin A-chain; relaxin
B-chain; prorelaxin; mouse gonadotropin-associated peptide; a
microbial protein, such as beta-lactamase; DNase; inhibin; activin;
vascular endothelial growth factor (VEGF); receptors for hormones
or growth factors; integrin; protein A or D; rheumatoid factors; a
neurotrophic factor such as brain-derived neurotrophic factor
(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6),
or a nerve growth factor such as NGF-.beta.; cardiotrophins
(cardiac hypertrophy factor) such as cardiotrophin-1 (CT-1);
platelet-derived growth factor (PDGF); fibroblast growth factor
such as aFGF and bFGF; epidermal growth factor (EGF); transforming
growth factor (TGF) such as TGF-alpha and TGF-beta, including
TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, or TGF-.beta.5;
insulin-like growth factor-I and -II (IGF-I and IGF-II);
des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding
proteins; CD proteins such as CD-3, CD-4, CD-8, and CD-19;
erythropoietin; osteoinductive factors; immunotoxins; a bone
morphogenetic protein (BMP); an interferon such as
interferon-alpha, -beta, and -gamma; colony stimulating factors
(CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g.,
IL-1 to IL-10; anti-HER-2 antibody; superoxide dismutase; T-cell
receptors; surface membrane proteins; decay accelerating factor;
viral antigen such as, for example, a portion of the AIDS envelope;
transport proteins; homing receptors; addressins; regulatory
proteins; antibodies; and fragments of any of the above-listed
polypeptides.
The preferred exogenous polypeptides of interest are mammalian
polypeptides. Examples of such mammalian polypeptides include t-PA,
gp120, anti-HER-2, DNase, IGF-I, IGF-II, brain IGF-I, growth
hormone, relaxin chains, growth hormone releasing factor, insulin
chains or pro-insulin, urokinase, immunotoxins, neurotrophins, and
antigens. Particularly preferred mammalian polypeptides include,
e.g., t-PA, gp120 (IIIb), anti-HER-2, DNase, IGF-I, IGF-II, growth
hormone, NGF, NT-3, NT4, NT-5, and NT-6, more preferably IGF, most
preferably IGF-I, and the most preferred mammalian polypeptide is a
human polypeptide.
As used herein, "IGF-I" refers to insulin-like growth factor from
any species, including bovine, ovine, porcine, equine, and
preferably human, in native sequence or in variant form and
recombinantly produced. In a preferred method, the IGF-I is cloned
and its DNA expressed, e.g., by the process described in EP 128,733
published Dec. 19, 1984.
The expression "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for bacteria include the alkaline phosphatase promoter,
optionally an operator sequence, and a ribosome-binding site.
Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist, the synthetic oligonucleotide adaptors or
linkers are used in accordance with conventional practice.
As used herein, the expressions "cell," "cell line," and "cell
culture" are used interchangeably and all such designations include
progeny. Thus, the words "transformants" and "transformed cells"
include the primary subject cell and cultures derived therefrom
without regard for the number of transfers. It is also understood
that all progeny may not be precisely identical in DNA content, due
to deliberate or inadvertent mutations. Mutant progeny that have
the same function or biological activity as screened for in the
originally transformed cell are included. Where distinct
designations are intended, it will be clear from the context.
The technique of "polymerase chain reaction," or "PCR," as used
herein generally refers to a procedure wherein minute amounts of a
specific piece of nucleic acid, RNA, and/or DNA, are amplified as
described in U.S. Pat. No. 4,683,195 issued 28 Jul. 1987.
Generally, sequence information from the ends of the region of
interest or beyond needs to be available, such that oligonucleotide
primers can be designed; these primers will be identical or similar
in sequence to opposite strands of the template to be amplified.
The 5'-terminal nucleotides of the two primers may coincide with
the ends of the amplified material. PCR can be used to amplify
specific RNA sequences, specific DNA sequences from total genomic
DNA, and cDNA transcribed from total cellular RNA, bacteriophage or
plasmid sequences, etc. See generally Mullis et al., Cold Spring
Harbor Symp. Quant. Biol., 51: 263 (1987); Erlich, ed., PCR
Technology, (Stockton Press, New York, 1989). For a recent review
on PCR advances, see Erlich et al., Science, 252: 1643-1650
(1991).
As used herein, PCR is considered to be one, but not the only,
example of a nucleic acid polymerase reaction method for amplifying
a nucleic acid test sample comprising the use of a known nucleic
acid as a primer and a nucleic acid polymerase to amplify or
generate a specific piece of nucleic acid.
B. Modes for Carrying Out the Invention
In the process herein, expression of the dsbA or dsbC gene is
induced just before (immediately prior to) heterologous gene
expression. The heterologous polypeptide and DsbA or DsbC protein
are both secreted into the periplasm or the heterologous
polypeptide is secreted into the culture medium of the bacteria
into which nucleic acid encoding these polypeptides has been
introduced. Preferably, the polypeptide is recovered from the
periplasm of the bacteria.
The dsbA or dsbC nucleic acid may be eDNA or genomic DNA from any
bacterial source, and is generally the native sequence. It is
suitably separately placed from the nucleic acid encoding the
heterologous polypeptide if nucleic acids are on the same vector,
i.e., they are not linked. In addition, the nucleic acid encoding
DsbA or DsbC and the nucleic acid encoding the heterologous
polypeptide are under separate, different inducible promoters so
that induction of expression can occur in the required sequential
order. The nucleic acid encoding the DsbA or DsbC and the nucleic
acid encoding the heterologous polypeptide may be integrated into
the host cell genome or contained on autonomously replicating
plasmids.
If the DsbA or DsbC are native products of the host cell, and if
the factors controlling expression of these native genes are
understood, such factors can be manipulated to achieve
over-expression of the dsbA or dsbC genes, e.g., by induction of
transcription from the natural promoter using known inducer
molecules, by mutation of the nucleic acids controlling or
repressing expression of the gene product to produce a mutant
strain that inductively over-expresses the gene product, by second
site mutations which depress the synthesis or function of factors
that normally repress the transcription of the gene product, and
the like.
In one alternative, the bacteria comprises two separate vectors
respectively containing the nucleic acid encoding the DsbA or DsbC
protein and the nucleic acid encoding the heterologous
polypeptide.
In another alternative, the nucleic acid encoding the DsbA or DsbC
protein and the nucleic acid encoding the heterologous polypeptide
are contained on the same vector but are under the control of
separate inducible promoters and separate signal sequences.
The heterologous nucleic acid (e.g., cDNA or genomic DNA) is
suitably inserted into a replicable vector for expression in the
bacterium under the control of a suitable promoter for bacteria.
Many vectors are available for this purpose, and selection of the
appropriate vector will depend mainly on the size of the nucleic
acid to be inserted into the vector and the particular host cell to
be transformed with the vector. Each vector contains various
components depending on its function (amplification of DNA or
expression of DNA) and the particular host cell with which it is
compatible. The vector components for bacterial transformation
generally include, but are not limited to, one or more of the
following: a signal sequence, an origin of replication, one or more
marker genes, and an inducible promoter.
In general, plasmid vectors containing replicon and control
sequences that are derived from species compatible with the host
cell are used in connection with bacterial hosts. The vector
ordinarily carries a replication site, as well as marking sequences
that are capable of providing phenotypic selection in transformed
cells. For example, E. coli is typically transformed using pBR322,
a plasmid derived from an E. coli species (see, e.g., Bolivar et
al., Gene, 2: 95 [1977]). pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR322 plasmid, or other
microbial plasmid or phage, also generally contains, or is modified
to contain, promoters that can be used by the microbial organism
for expression of the selectable marker genes.
The DNA encoding the polypeptide of interest herein may be
expressed not only directly, but also as a fusion with another
polypeptide, preferably a signal sequence or other polypeptide
having a specific cleavage site at the N-terminus of the mature
polypeptide. In general, the signal sequence may be a component of
the vector, or it may be a part of the polypeptide DNA that is
inserted into the vector. The heterologous signal sequence selected
should be one that is recognized and processed (i.e., cleaved by a
signal peptidase) by the host cell. For bacterial host cells that
do not recognize and process the native polypeptide signal
sequence, the signal sequence is substituted by a bacterial signal
sequence selected, for example, from the group consisting of the
alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders.
Both expression and cloning vectors contain a nucleic acid sequence
that enables the vector to replicate in one or more selected host
cells. Generally, in cloning vectors this sequence is one that
enables the vector to replicate independently of the host
chromosomal DNA, and includes origins of replication or
autonomously replicating sequences. Such sequences are well known
for a variety of bacteria. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria.
Expression and cloning vectors also generally contain a selection
gene, also termed a selectable marker. This gene encodes a protein
necessary for the survival or growth of transformed host cells
grown in a selective culture medium. Host cells not transformed
with the vector containing the selection gene will not survive in
the culture medium. Typical selection genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g.,
ampicillin, neomycin, methotrexate, or tetracycline, (b) complement
auxotrophic deficiencies, or (c) supply critical nutrients not
available from complex media, e.g., the gene encoding D-alanine
racemase for Bacilli. One example of a selection scheme utilizes a
drug to arrest growth of a host cell. Those cells that are
successfully tramformed with a heterologous gene produce a protein
conferring drug resistance and thus survive the selection
regimen.
The expression vector for producing a heterologous polypeptide also
contains an inducible promoter that is recognized by the host
bacterial organism and is operably linked to the nucleic acid
encoding the polypeptide of interest. It also contains a separate
inducible promoter operably linked to the nucleic acid encoding the
DsbA or DsbC protein. Inducible promoters suitable for use with
bacterial hosts include the .beta.-lactamase and lactose promoter
systems (Chang et al., Nature, 275: 615 [1978]; Goeddel et al.,
Nature, 281: 544 [1979]), the arabinose promoter system (Guzman et
al., J. Bacteriol., 174: 7716-7728 [1992]), alkaline phosphatase, a
tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:
4057 [1980] and EP 36,776) and hybrid promoters such as the tac
promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25
[1983]). However, other known bacterial inducible promoters are
suitable. Their nucleotide sequences have been published, thereby
enabling a skilled worker operably to ligate them to DNA encoding
the polypeptide of interest or to the dsbA or dsbC genes
(Siebenlist et al., Cell, b 20: 269 [1980]) using linkers or
adaptors to supply any required restriction sites.
Promoters for use in bacterial systems also generally contain a
Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding
the polypeptide of interest. The promoter can be removed from the
bacterial source DNA by restriction enzyme digestion and inserted
into the vector containing the desired DNA.
Construction of suitable vectors containing one or more of the
above-listed components employs standard ligation techniques.
Isolated plasmids or DNA fragments are cleaved, tailored, and
re-ligated in the form desired to generate the plasmids
required.
For analysis to confirm correct sequences in plasmids constructed,
the ligation mixtures are used to transform E. coli K12 strain 294
(ATCC 31,446) or other strains, and successful transformants are
selected by ampicillin or tetracycline resistance where
appropriate. Plasmids from the transformants are prepared, analyzed
by restriction endonuclease digestion, and/or sequenced by the
method of Sanger et al., Proc. Natl. Acad. Sci. USA, 74: 5463-5467
(1977) or Messing et al., Nucleic Acids Res., 9: 309 (1981) or by
the method of Maxam et al., Methods in Enzymology, 65: 499
(1980).
Suitable bacteria for this purpose include archaebacteria and
eubacteria, especially eubacteria, and most preferably
Enterobacteriaceae. Examples of useful bacteria include
Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus,
Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella,
Rhizobia, Vitreoscilla, and Paracoccus. Suitable E. coli hosts
include E. coli W3110 (ATCC 27,325), E. coli 294 (ATCC 31,446), E.
coli B, and E. coli X1776 (ATCC 31,537). These examples are
illustrative rather than limiting. Mutant cells of any of the
above-mentioned bacteria may also be employed. It is, of course,
necessary to select the appropriate bacteria taking into
consideration replicability of the replicon in the cells of a
bacterium. For example, E. coli, Serratia, or Salmonella species
can be suitably used as the host when well known plasmids such as
pBR322, pBR325, pACYC177, or pKN410 are used to supply the
replicon.
E. coli strain W3110 is a preferred host or parent host because it
is a common host strain for recombinant DNA product fermentations.
Preferably, the host cell should secrete minimal amounts of
proteolytic enzymes. For example, strain W3110 may be modified to
effect a genetic mutation in the genes encoding proteins, with
examples of such hosts including E. coli W3110 strain 1A2, which
has the complete genotype tonA.DELTA.; E. coli W3110 strain 9E4,
which has the complete genotype tonA.DELTA. ptr3; E. coli W3110
strain 27C7 (ATCC 55,244), which has the complete genotype
tonA.DELTA. ptr3 phoA.DELTA.E15 .DELTA.(argF-lac)169
ompT.DELTA..alpha. degP41kan.sup.r ; E. coli W3110 strain 37D6,
which has the complete genotype tonA.DELTA. ptr3 phoA.DELTA.E15
.DELTA.(argF-lac)169 ompT.DELTA..alpha. degP41kan.sup.r rbs7.DELTA.
ilvG; E. coli W3110 strain 40B4, which is strain 37D6 with a
non-kanamycin resistant degP deletion mutation; E. coli W3110
strain 33D3, which has the complete genotype tonA ptr3 lacIq LacL8
ompT.DELTA.degP kan.sup.r ; E. coli W3110 strain 36F8, which has
the complete genotype tonA phoA.DELTA. (argF-lac) ptr3 degP
kan.sup.R ilvG+, and is temperature resistant at 37.degree. C.; and
an E. coli strain having the mutant periplasmic protease(s)
disclosed in U.S. Pat. No. 4,946,783 issued Aug. 7, 1990.
Host cells are transfected and preferably transformed with the
above-described expression vectors and cultured in conventional
nutrient media modified as appropriate for inducing promoters,
selecting transformants, or amplifying the genes encoding the
desired sequences.
Transfection refers to the taking up of an expression vector by a
host cell whether or not any coding sequences are in fact
expressed. Numerous methods of transfection are known to the
ordinarily skilled artisan, for example, CaPO.sub.4 and
electroporation. Successful transfection is generally recognized
when any indication of the operation of this vector occurs within
the host cell.
Transformation means introducing DNA into an organism so that the
DNA is replicable, either as an extrachromosomal element or by
chromosomal integrant. Depending on the host cell used,
transformation is done using standard techniques appropriate to
such cells. The calcium treatment employing calcium chloride, as
described in section 1.82 of Sambrook et al., Molecular Cloning: A
Laboratory Manual [New York: Cold Spring Harbor Laboratory Press,
1989], is generally used for bacterial cells that contain
substantial cell-wall barriers. Another method for transformation
employs polyethylene glycol/DMSO, as described in Chung and Miller,
Nucleic Acids Res., 16: 3580 (1988). Yet another method is the use
of the technique termed electroporation.
Bacterial cells used to produce the polypeptide of interest for
purposes of this invention are cultured in suitable media in which
the promoters for the nucleic acid encoding the heterologous
polypeptide and for the nucleic acid encoding the DsbA or DsbC can
be artificially induced as described generally, e.g., in Sambrook
et al., supra. Examples of suitable media are given in U.S. Pat.
Nos. 5,304,472 and 5,342,763.
Any necessary supplements besides carbon, nitrogen, and inorganic
phosphate sources may also be included at appropriate
concentrations introduced alone or as a mixture with another
supplement or medium such as a complex nitrogen source. The pH of
the medium may be any pH from about 5-9, depending mainly on the
host organism. Preferably, the medium contains no reduced
glutathione, and the bacteria are not cultured so as to
over-express nucleic acid encoding the heat-shock transcription
factor, RpoH.
For induction, typically the cells are cultured until a certain
optical density is achieved, e.g., a A.sub.550 of about 60-80, at
which point induction is initiated (e.g., by addition of an
inducer, by depletion of a medium component, etc.), to induce
expression of the dsbA or dsbC gene. When the optical density
reaches a higher amount, e.g., a A.sub.55 of about 80-100,
induction of the second promoter for the heterologous polypeptide
is effected.
Gene expression may be measured in a sample directly, for example,
by conventional northern blotting to quantitate the transcription
of mRNA. Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5205 (1980).
Various labels may be employed, most commonly radioisotopes,
particularly .sup.32 P. However, other techniques may also be
employed, such as using biotin-modified nucleotides for
introduction into a polynucleotide. The biotin then serves as the
site for binding to avidin or antibodies, which may be labeled with
a wide variety of labels, such as radionuclides, fluorescers,
enzymes, or the like.
Procedures for observing whether an expressed or over-expressed
gene product is secreted are readily available to the skilled
practitioner. Once the culture medium is separated from the host
cells, for example, by centrifugation or filtration, the gene
product can then be detected in the cell-free culture medium by
taking advantage of known properties characteristic of the gene
product. Such properties can include the distinct immunological,
enzymatic, or physical properties of the gene product.
For example, if an over-expressed gene product has a unique enzyme
activity, an assay for that activity can be performed on the
culture medium used by the host cells. Moreover, when antibodies
reactive against a given gene product are available, such
antibodies can be used to detect the gene product in any knon
immunological assay (e.g., as in Hadowe et al., Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, New York,
1988).
The secreted gene product can also be detected using tests that
distinguish polypeptides on the basis of characteristic physical
properties such as molecular weight. To detect the physical
properties of the gene product, all polypeptides newly synthesized
by the host cell can be labeled, e.g., with a radioisotope. Common
radioisotopes that can be used to label polypeptides synthesized
within a host cell include tritium (.sup.3 H), carbon-14 (.sup.14
C), sulfur-35 (.sup.35 S), and the like. For example, the host cell
can be grown in .sup.35 S-methionine or .sup.35 S-cysteine medium,
and a significant amount of the .sup.35 S label will be
preferentially incorporated into any newly synthesized polypeptide,
including the over-expressed heterologous polypeptide. The .sup.35
S-containing culture medium is then removed and the cells are
washed and placed in fresh non-radioactive culture medium. After
the cells are maintained in the fresh medium for a time and under
conditions sufficient to allow secretion of the .sup.35
S-radiolabeled expressed heterologous polypeptide, the culture
medium is collected and separated from the host cells. The
molecular weight of the secreted, labeled polypeptide in the
culture medium can then be determined by known procedures, e.g.,
polyacrylamide gel electrophoresis. Such procedures, and/or other
procedures for detecting secreted gene products, are provided in
Goeddel, D. V. (ed.) 1990, Gene Expression Technology, Methods in
Enzymology, Vol. 185 (Academic Press), and Sambrook et al.,
supra.
For secretion of an expressed or over-expressed gene product, the
host cell is cultured under conditions sufficient for secretion of
the gene product. Such conditions include, e.g., temperature,
nutrient, and cell density conditions that permit secretion by the
cell. Moreover, such conditions are those under which the cell can
perform basic cellular functions of transcription, translation, and
passage of proteins from one cellular compartment to another, as
are known to those skilled in the art.
In practicing the process of this invention, the yield of total
polypeptide is generally increased, while yield of soluble
polypeptide is not changed or is decreased, i.e., yield of
insoluble (aggregated) polypeptide is increased.
The polypeptide of interest is recovered from the periplasm or
culture medium as a secreted polypeptide. It is often preferred to
purify the polypeptide of interest from recombinant cell proteins
or polypeptides and from the DsbA or DsbC protein to obtain
preparations that are substantially homogeneous as to the
polypeptide of interest. As a first step, the culture medium or
lysate is centrifuged to remove particulate cell debris. The
membrane and soluble protein fractions may then be separated if
necessary. The polypeptide may then be purified from the soluble
protein fraction and from the membrane fraction of the culture
lysate, depending on whether the polypeptide is membrane bound, is
soluble, or is present in an aggregated form. The polypeptide
thereafter is solubilized and refolded, if necessary, and is
purified from contaminant soluble proteins and polypeptides. In a
preferred embodiment, the aggregated polypeptide is isolated,
followed by a simultaneous solubilization and refolding step, as
disclosed in U.S. Pat. No. 5,288,931.
Another preferred method for isolating exogenous polypeptides from
a complex biological mixture containing polypeptides and
non-polypeptides contained in a fermentation broth, such as the one
described above, involves contact of reagents with the cells,
preferably the cell culture, containing the polypeptide in a
non-native conformation, so that an aqueous extraction/isolation
can take place. Preferably, the method entails direct addition of
reagents to the fermentation vessel after the polypeptide has been
produced recombinantly, thereby avoiding extra steps of harvesting,
homogenization, and centrifugation to obtain the polypeptide. While
the remaining particulates can be removed by Gaulin homogenization
and re-suspension, filtration, or a combination thereof, this
method utilizes a multiple-phase extraction system for purifying
recombinant polypeptides from the remaining particulates.
In particular, this method is preferred for non-native mammalian
polypeptides produced recombinantly in bacterial cells, including
E. coli, which form refractile bodies in the periplasm of the
cells. In this system, one or more denaturants (chaotropic agent),
such as urea, guanidine hydrochloride, and/or a base, and a
reducing agent, such as dithiothreitol or cysteine, are added to
the polypeptide-containing medium and then phase-forming species
are added to the broth. Once this second group of reagents is added
to the broth, multiple phases are formed whereby one phase is
enriched in the polypeptide and depleted in biomass solids and
nucleic acids. Preferably, the system has two to four phases, and
more preferably two phases, one being enriched in polypeptide and
the other being enriched in biomass solids and nucleic acids.
Preferably, the desired polypeptide partitions to the upper phase
so that the upper phase is enriched in the polypeptide and depleted
in the biomass solids and nucleic acids.
Specifically, after fermentation is complete, the cell culture is
contacted with one or more chaotropic agents, an optional reducing
agent, and phase-forming reagents so that multiple phases are
formed, one phase of which is enriched in the polypeptide of
interest. It is preferred to add the chaotrope and reducing agent
first to extract the polypeptide from the cell and maintain its
solubility in the broth before the phase-forming reagents are
added. Also, while the polypeptide of interest can be extracted
from (and enriched in) any phase, preferably it is recovered from
the uppermost phase.
Most preferably, the chaotropic agent and optional reducing agent
are added directly to the fermentation broth in the fermentation
vessel before isolation of the polypeptide so that the reagents
permeate the cells and the polypeptide is solubilized and diffuses
to the surrounding medium. The reducing agent is added if the
polypeptide contains at least one sulfhydryl group.
Examples of suitable reducing agents include dithiothreitol
(DTT),/.beta.-mercaptoethanol (BME), cysteine, thioglycolate, and
sodium borohydride. The amount of reducing agent to be present in
the buffer will depend mainly on the type of reducing agent and
chaotropic agent, the type and pH of the buffer employed, and the
type and concentration of the polypeptide in the buffer. An
effective amount of reducing agent is that which is sufficient to
eliminate intermolecular disulfide-mediated aggregation. For
example, with 0.5-6 mg/mL IGF-I in a buffered solution at pH
7.5-10.5 containing 1-4M urea, the DTT concentration is at about
1-20 mM, and the concentration of cysteine is at about 10-50 mM.
The preferred reducing agent is DTT at about 2-10 mM or cysteine at
about 30-50 mM.
Chaotropic agents suitable for practicing this method of extraction
include, e.g., urea and salts of guanidine or thiocyanate, more
preferably urea, guanidine hydrochloride, or sodium thiocyanate.
The amount of chaotropic agent necessary to be present in the
buffer depends, for example, on the type of chaotropic agent and
polypeptide present. The amount of chaotropic agent to be added to
the fermentation broth will be sufficiently high to extract the
polypeptide from the cell and maintain its solubility in the broth.
If the polypeptide is to be extracted from the top phase, the
amount of chaotropic agent must be sufficiently low so that after
addition of the phase-forming species, the density is not increased
to a point where the solids rise to the top instead of settling to
the bottom. Generally the concentration of chaotropic agent is
about 0.1 to 9M, preferably about 0.5-9M, more preferably about 0.5
to 6M, and most preferably about 0.5-3M. Also, preferably the
chaotropic agent is added to the culture medium before the
phase-forming reagents are added. The preferred chaotropic agent
herein is urea at about 1.5-2.5M, more preferably at about 2M, or
guanidine hydrochloride at about 0.5-3M. Most preferably, the
chaotropic agent is urea.
The concentration of the polypeptide in the aqueous solution to
which the chaotrope and reducing agent are added must be such that
the polypeptide will be recovered in the maximum yield. The exact
amount to employ will depend, e.g., on the type of polypeptide and
the concentrations and types of other ingredients in the aqueous
solution, particularly the reducing agent, chaotropic agent,
phase-forming species, and pH. For polypeptides in general, the
preferred concentration of polypeptide is about 0.1 to 15 mg/mL.
The preferred concentration of IGF-I (resulting in the maximum
yield of denatured or non-native IGF-I) is in the range of 0.5-6 mg
per mL, more preferably 1.5-5 mg/mL.
The types of phase-forming species to employ herein depend on many
factors, including the type of polypeptide and the ingredients in
the fermentation broth being treated. The species must be selected
so that the polypeptide does not precipitate and one phase is more
hydrophobic than the other phase so that the polypeptide will be
located in the more hydrophobic phase and the biomass solids and
nucleic acids will settle to the less hydrophobic phase.
The phase-forming species may be a combination of agents, including
polymer combinations (polymer-polymer), polymer-salt combinations,
solvent-salt combinations, and polymer-solvent combinations.
Suitable polymers are both highly hydrophilic polymers and less
hydrophilic polymers, i.e., any phase-forming polymers that are
known in the art. Examples include polyethylene glycol or
derivatives thereof, including various molecular weights of PEG
such as PEG 4000, PEG 6000, and PEG 8000, derivatives of PEG
described, for example, in Grunfeld et al., Appl. Biochem.
Biotechnol., 33: 117-138 (1992), polyvinylpyrrolidone (PVP), in a
preferable molecular weight range of about 36,000 to 360,000,
starches such as dextran (e.g., dextran 70 and 500), dextrins, and
maltodextrins (preferable molecular weight between about 600 and
5,000), sucrose, and Ficoll-400.TM. polymer (a copolymer of sucrose
and epichlorohydrin). The preferred polymer herein is polyethylene
glycol, polypropylene glycol, polyvinylpyrrolidone, or a
polysaccharide such as a dextran. The most preferred polymer herein
is PEG of different molecular weights or a PEG-polypropylene glycol
combination or copolymer.
Examples of suitable organic solvents include ethylene glycol,
glycerol, dimethyl sulfoxide, polyvinylalcohol, dimethylformamide,
dioxane, and alcohols such as methanol, ethanol, and 2-propanol.
Such solvents are such that, when added to aqueous solution, they
increase the hydrophobicity of the solution.
The salts can be inorganic or organic and preferably do not act to
precipitate the polypeptide. Salts containing transition elements
are not preferred as they tend to precipitate the polypeptide.
Anions are selected that have the potential for forming aqueous
multiple-phase systems. Examples include ammonium sulfate, sodium
dibasic phosphate, sodium sulfate, ammonium phosphate, potassium
citrate, magnesium phosphate, sodium phosphate, calcium phosphate,
potassium phosphate, potassium sulfate, magnesium sulfate, calcium
sulfate, sodium citrate, manganese sulfate, manganese phosphate,
etc. Types of salts that are useful in forming bi-phasic aqueous
systems are evaluated more fully in Zaslavskii et al., J. Chrom.,
439: 267-281 (1988). Preferred salts herein are sulfates,
phosphates, or citrates and are alkali or alkaline earth metals.
More preferred are sulfates and citrates, and most preferred are
sulfates since there are fewer pH limitations with sulfates. The
most preferred salts herein are sodium sulfate and sodium
citrate.
The amounts of phase-forming species to add to the polypeptide of
interest to obtain a satisfactory multiple-phase system are those
known in the art. The amount of phase-forming species added to the
polypeptide will depend on such factors as, for example, the amount
of chaotropic agent and reducing agent, if any, already present in
the fermentation broth, the nature of the cell culture media, the
type of cells used in the fermentation, the type of polypeptide
being treated, whether the polypeptide will be recovered from the
lower or upper phase, and the type(s) of phase-forming species
being added. The general concentration of polymer employed is about
5% (w/w) up to the limit of solubility for the polymer and the
concentration of salt employed is about 3% (w/w) up to the limit of
solubility for the salt, depending on the size of the phase-volume
ratio needed. The phase-volume ratio must be sufficient to
accommodate the biomass solids. The types and amounts of
phase-forming species that are effective can be determined by phase
diagrams and by evaluating the final result, i.e., the degree of
purity and the yield of the polypeptide of interest. If the
phase-forming species are a polymer-salt combination, preferably
the concentration of salt added is about 4-15% (w/w) and the
concentration of polymer is 5-18% (w/w) so that the desired
polypeptide will be in an opposite phase from that in which the
biomass solids and nucleic acids are present.
If the system desired is one where the polypeptide is distributed
in the top phase and the biomass solids and nucleic acids are in
the bottom phase, then there is a window of concentrations of
phase-forming species. When higher amounts of chaotropic agent are
added to maintain solubilization, the higher the amount of
phase-forming species required. However, a high concentration of
all these reagents will increase the density of the solution. A
high density will cause the biomass solids to settle less readily.
An overly high density will cause biomass solids to float on the
surface. Hence, the concentrations of chaotropic agent and
phase-forming species must be sufficiently high to maintain a fully
solubilized polypeptide, but low enough to allow the biomass solids
to sediment to the opposite (lower) phase.
If the polypeptide is to be recovered in the upper phase, typically
the salt concentration will be about 4-7% (w/w) and the polymer
concentration will be about 12-18% (w/w), depending, e.g., on the
type of salt, polymer, and polypeptide. If an organic solvent is
added as a phase-forming species, such as ethanol, it is preferably
added in a concentration of about 10 to 30% (volume/volume) of the
solution, depending, e.g., on the type of polypeptide and alcohol
and if any other phase-forming species is present, preferably at a
concentration of about 20% (v/v).
The exact conditions for contacting the cell culture with the
various reagents will depend on, e.g., the pH of the buffer, the
types of phase-forming reagents, and the types and concentrations
of polypeptide and chaotropic and reducing agents. The reaction
temperature is generally about 20.degree.-40.degree. C., more
preferably room temperature. The contacting step will generally be
carried out for at least about 30 minutes, preferably about 30
minutes to 12 hours depending on whether side-reactions will occur,
more preferably about 30 minutes to 8 hours, and most preferably
about 30 minutes to 1.5 hours.
If the polypeptide is being unfolded, the degree of unfolding is
suitably determined by chromatography of the non-native
polypeptide, including hydrophobic interaction chromatography or
ion-exchange chromatography. Increasing peak area for the
non-native material indicates how much nonnative polypeptide is
present.
Once the multiple-phase system is established, one phase will be
enriched in the polypeptide and depleted in the disrupted particles
and cells comprising the biomass solids and nucleic acids. In a
two-phase system, preferably the top phase is enriched in the
polypeptide whereas the bottom phase is enriched in the disrupted
particles and cells. The polypeptide can be easily recovered by
separation of the phases. This recovery step may be accomplished by
decanting the upper phase, by draining the lower phase, or by
centrifugation. The polypeptide can then be isolated from the phase
in which it is contained by changing the pH of the phase so as to
precipitate the polypeptide or by adding a suitable solvent,
whereupon the precipitated polypeptide is suitably recovered by
centrifugation or filtration or as a slurry. Alternatively, the
polypeptide can be recovered from the polymer-containing phase by
re-extraction by addition of a suitable polymer, salt, or solvent.
In the case of IGF-I, the polypeptide is recovered from the
isolated polymer phase by lowering the pH so that the IGF-I will
precipitate, resulting in a yield of IGF-I of as much as or more
than about 97%. The DsbA or DsbC protein would be separated from
the polypeptide at this stage.
Once obtained from the liquid phase of the multiple-phase system,
or at a later stage of purification, the polypeptide is suitably
refolded into an active conformation. One suitable refolding method
that can be utilized is that which follows.
After the polypeptide is solubilized and extracted by the
multiple-phase extraction system herein, it is placed or diluted
into a buffer containing solvent, chaotropic agent, salt, and a
minimal amount of a copper or manganese salt. This buffer
unexpectedly increases refolding yields of polypeptide from any
type of host. This buffer is at a pH of about 7 to 12, depending
mainly on the type of polypeptide and reducing agent, preferably
about 8 to 11, more preferably pH 8.5 to 11, and most preferably
8.5 to 10.5.
One key ingredient of the buffer is an alcoholic or polar aprotic
solvent at a concentration of about 5-40% (v/v), preferably 10 to
30% (volume/volume) of the solution, depending, e.g., on the type
of polypeptide and solvent, and the concentration of chaotropic
agent. It is most preferably at a concentration of about 20%
(v/v).
A second key ingredient to this buffer is an alkaline earth, alkali
metal, or ammonium salt, which is present in a concentration of
about 0.2 to 3M, preferably 0.2 to 2M, depending mainly on the
chaotrope concentration, solvent concentration, and the type of
alkaline earth, alkali metal, or ammonium salt and polypeptide
employed. For example, if the cation is sodium, potassium, or
ammonium, the concentration is about 0.5 to 3M, but if the cation
is magnesium, the concentration is about 0.2 to
A third key ingredient of the buffer is effective amount of a
chaotropic agent. The amount of such chaotrope will depend mainly
on the concentration of alkaline earth, alkali metal, or ammonium
salt, the concentration of solvent, the specific type of alkaline
earth, alkali metal, or ammonium salt employed, the specific type
of chaotropic agent employed, and the type of polypeptide, as well
as the pH of the buffer, but in general will range from about 0.1
to 9M, preferably about 0.5 to 6M, and most preferably about 1.5 to
4M. As to specific chaotropes, preferably about 0.1 to 2M of
guanidine hydrochloride, and preferably about 1-3M, more preferably
about 1-2.5M, and most preferably about 2M, of urea is
utilized.
A fourth key ingredient of the buffer is an effective amount of a
transition metal salt selected from copper and manganese salts so
that oxidation and resultant refolding will occur. The amount of
copper or manganese salt depends mainly on the type of transition
metal and polypeptide employed and the oxygen level present. The
lower the rate of oxygen addition or the oxygen level, the higher
the amount of copper or manganese salt that can be employed. The
copper or manganese salt concentration is typically about 0.01 to
15 .mu.M, preferably about 0.01 to 10 .mu.M, more preferably about
0.01 to 5 .mu.M, and even more preferably about 0.01 to 0.5 .mu.M.
The above preferred ranges are particularly preferred for IGF-I. If
the concentration is increased beyond about 15 .mu.M, unexpectedly
the yield of correctly folded polypeptide decreases dramatically.
Most preferably, the concentration of a copper or manganese salt is
about 0.5 .mu.M. The transition metal salt may already be present
in the buffer without addition of exogenous transition metal salt,
for example, if it is residual from the fermentation, or it may be
added to the buffer, or both.
The buffer can be any of those listed above for the first buffered
solution, with CAPSO, glycine, and CAPS being preferred at pH
8.5-11, particularly at a concentration of about 20 mM, and most
preferably CAPSO and glycine. The polypeptide may be diluted with
the refolding buffer, preferably at least five fold, more
preferably at least about ten fold. Alternatively, the polypeptide
may be dialyzed against the refolding buffer. The refolding is
typically carried out at about 0.degree.-45.degree. C., preferably
about 20.degree.-40.degree. C., more preferably about
23.degree.-37.degree. C., even more preferably about
25.degree.-37.degree. C., and most preferably about 25.degree. C.
for at least about one hour. The preferred temperature is not
apparently affected by salt, solvent, and chaotropic agent levels,
but may be affected by the presence of sucrose and glycerol, in
which case it should be kept above about 20.degree. C. The solution
optionally also contains a reducing agent and an osmolyte.
The reducing agent is suitably selected from those described above
for the solubilizing step in the concentration range given. Its
concentration will depend especially on the concentrations of
alkaline earth, alkali metal, or ammonium salt, polypeptide, and
solvent. Preferably, the concentration of reducing agent is about
0.5 to 8 mM, more preferably about 1-5 mM, even more preferably
about 0.5-2 mM. The preferred reducing agents are DTT and
cysteine.
The optional osmolyte is preferably sucrose (in a concentration of
about 0.25-1M) or glycerol (in a concentration of about 1-14M).
More preferably, the sucrose concentration is at about 1M and the
glycerol concentration is at about 4M.
The initial concentration of polypeptide in the folding buffer is
such that the ratio of correctly folded to misfolded conformer
recovered will be maximized, as determined by HPLC, RIA, or
bioassay. The exact concentration will depend, for example, on the
type of polypeptide employed. The preferred concentration of
polypeptide (resulting in the maximum yield of correctly folded
conformer) is in the range of about 0.1 to 15 mg/mL, more
preferably about 0.1 to 6 mg/mL, and most preferably about 0.2 to 5
mg/mL.
In addition, a source of oxygen such as air or oxygen gas is
entrained in or otherwise introduced into the buffer so as to
effect oxidation together with the copper or manganese salt. The
oxygen can be present in the buffer at any point in time, including
before the polypeptide or any other reagents are added to the
buffer.
The amount of oxygen source introduced will depend, e.g., on the
type of vessel utilized, the type and concentration of polypeptide,
the type of oxygen source, the type and amount of copper or
manganese salt, and the type and amount of reducing agent present,
if any, and the type and amount of chaotropic agent present as well
as the pH of the buffer. Generally, the oxygen source will be
introduced by passive means (e.g., as air in head space in a ratio
of air space to fluid volume of 2:1) using an agitator.
Alternatively, the oxygen source may be introduced by bubbling
through a sparger. The rate of introduction of the oxygen must be
sufficient to allow folding to reach completion in preferably about
1 to 12 hours, more preferably about 1 to 6 hours, and most
preferably about 1 to 3 hours. The addition of molar oxygen is
proportional to the reductant concentration and polypeptide
concentration, but inversely proportional to the copper or
magnesium salt concentration. The rate of oxidation is limited by
the level of catalyst, not by the oxygen addition rate. A higher
sparging rate is required for larger volume folding.
The degree of refolding that occurs upon this second incubation is
suitably determined by the RIA titer of the polypeptide or by HPLC
analysis using e.g., a Vydac or Baker C-18 column, with increasing
RIA titer or correctly folded polypeptide peak size directly
correlating with increasing amounts of correct, biologically active
polypeptide conformer present in the buffer. The incubation is
carried out to maximize the yield of correctly folded polypeptide
conformer and the ratio of correctly folded polypeptide conformer
to misfolded polypeptide conformer recovered, as determined by RIA
or HPLC, and to minimize the yield of multimeric, associated
polypeptide as determined by mass balance.
The following procedures are exemplary of suitable purification
procedures: fractionation on immunoaffinity or ion-exchange
columns; ethanol precipitation; reverse phase HPLC; chromatography
on silica or on a cation-exchange resin such as DEAE;
chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; and gel
filtration using, for example, Sephadex G-75.
The following examples are offered by way of illustration and not
by way of limitation. The disclosures of all patent and scientific
references cited in the specification are expressly incorporated
herein by reference.
EXAMPLE I
The dsbA nucleotide sequence was amplified using PCR techniques
from p16-1 (Bardwell et al., supra, 1991) and a XbaI site was added
at the 5' end and a ClaI site at the 3' end of the DsbA-encoding
sequence. This DNA fragment was digested with the appropriate
restriction enzymes and ligated with a 1.0-kb PstI-XbaI fragment of
pKMTacII (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25
[1983]) and a 3.6-kb ClaI-PstI fragment of pBKIGF-2B (U.S. Pat. No.
5,342,763). The resulting plasmid, pJJ41 (see FIG. 1), contains
dsbA under the control of the tacII promoter and confers resistance
to ampicillin and tetracycline. This plasmid was digested with ClaI
and the ends were filled in by Klenow fragment and
deoxynucleotides.
After inactivation of the Klenow fragment and extraction of the
digested DNA, the DNA was subsequently digested with ScaI and the
1.5-kb fragment containing the 5' end of the .beta.-lactamase gene
and the TacII-dsbA element was purified. This was ligated with a
4.6-kb fragment of EcoRI-digested and filled-in pBKIGF-2B, which
had also been digested with ScaI. This fragment of pBKIGF-2 B
contained the 3' end of the .beta.-lactamase, allowing selection
for resistance to ampicillin as well as the DNA sequences for
resistance to tetracycline and production of IGF-I. This plasmid is
denoted pJJ42 (see FIG. 1).
This plasmid was transformed into E. coli strain 33D3 (with the
genotype W3110tonA ptr3 lacIq LacL8 ompT.DELTA.degP kan.sup.r. This
strain was constructed in several steps using techniques involving
transduction with phage P1kc, derived from P1 (J. Miller,
Experiments in Molecular Genetics), Cold Spring Harbor, New York,
Cold Spring Harbor Laboratory, 1972) and transposon genetics
Kleckner et al., J. Mol. Biol., 116: 125-159 [1977]). The starting
host used was E. coli K-12 W3110, which is a K-12 strain that is F-
lambda- (Bachmann, Bact. Rev., 36: 525-557 [1972]; Bachmann,
"Derivations and Genotypes of Some Mutant Derivatives of
Escherichia coli K-12," p. 1190-1219, in F. C. Neidhardt et al.,
ed., Escherichia coli and Salmonella typhimurium: Cellular and
Molecular Biology, vol. 2, American Society for Microbiology,
Washington, D.C., 1987).
Introduction of the tonA (fhuA) mutation and ptr3 mutation into the
genome is described in detail in U.S. Pat. No. 5,304,472 issued
Apr. 19, 1994. The resulting strain is designated 9E4. The
lacl.sup.q (Muller-Hill et al., Proc. Natl. Acad. Sci. USA, 59:
1259 [1968]) and lacL8 (Scaife and Beckwith, Cold Spring Harbor
Symp. Quant. Biol., 31: 403 [1967]) mutations were introduced into
strain 9E4 by co-transduction with proC. First, a linked Tn5
insertion into proC and a phoA.DELTA.E15 (Sarthy, A. et al., J.
Bacteriol., 145: 288-292 [1981]) mutation were introduced. P1
transduction to proline prototrophy restored the proC gene and the
phoA gene and also introduced the lacF.sup.q lacL8 mutations. The
lacI.sup.q mutation results in an overproduction of lac repressor
compared to wild-type. The presence of the lacI.sup.q mutation can
be observed on LB plates containing the chromogenic substrate
5-bromo-4-chloro-3 -indolyl-.beta.-D-galactoside (X-gal). On these
plates a lacI.sup.30 strain will be light blue, whereas a
lacI.sup.8 strain will be colorless. The LacL8 mutation is a
promoter mutation that results in lower lac operon enzyme levels.
The presence of the lacL8 mutation can be detected on MacConkey 1%
lactose medium as colonies with a dark red center and beige
surrounding edge.
Introduction of the ompT.DELTA.deletion and the degP mutation into
the strain is described in detail in U.S. Pat. No. 5,304,472.
The final strain 33D3 is resistant to T1 and .O slashed.80 phage,
lacks three proteases, overproduces the lac repressor, and produces
lower than wild-type lac operon enzyme levels when induced.
33D3 cells containing pJJ42 were then grown in a 10-liter fermentor
under conditions identical to IGF-I production as described in U.S.
Pat. No. 5,304,472, Example II, 1.times.feed conditions. When the
optical density (A.sub.550) of the culture reached 60, IPTG or
lactose was added in the amounts given below to induce the tacII
promoter, thereby increasing dsbA expression. The growth medium was
designed for phosphate depletion and phoA promoter induction to
occur at an optical density (A.sub.550) of 80 to 100. Total IGF-I
production was measured by a high-pressure liquid chromatography
(HPLC) assay described by U.S. Pat. No. 5,304,472.
The results are shown in Table 1 below:
TABLE 1 ______________________________________ Fermentation Run
Conditions g/L IGF-I ______________________________________ S1968
pBKIGF-2B, no inducer 3.0 S1963 pJJ42, 0.05 mM IPTG 4.8 S1964
pJJ42, 1.0 mm IPTG 4.8 S1959 pJJ42, 1.0% lactose 4.0
______________________________________
The results show that over-expression of the dsbA gene in the
periplasm of the bacteria improves yield of IGF-I for all rum that
were induced by either inducer. The increase in IGF-I accumulation
was about 60%. The correctly folded IGF-I can be recovered in high
yield by liquid-liquid extraction followed by aerobic refolding as
described above or by methods described in U.S. Pat. No.
5,288,931.
EXAMPLE II
The dsbC nucleotide sequence was amplified using PCR techniques
from the genome of E. coli (Missiakas et al., supra, [1994]) and a
XbaI site was added at the 5' end and a ClaI site at the 3' end of
the DsbC-encoding sequence. This DNA fragment was digested with the
appropriate restriction enzymes and ligated with a 1.0-kb PstI-XbaI
fragment of pKMTacII (deBoer et al., Proc. Natl. Acad. Sci. USA,
80: 21-25 [1983]) and a 3.6-kb ClaI-PstI fragment of pBKIGF-2B
(U.S. Pat. No. 5,342,763). The resulting plasmid, pJJ37 (see FIG.
2), contains dsbC under the control of the tacII promoter and
confers resistance to ampicillin and tetracycline. This plasmid was
digested with CtaI and the ends were filled in by Klenow fragment
and deoxynucleotides.
After inactivation of the Klenow fragment and extraction of the
digested DNA, the DNA was subsequently digested with ScaI and the
1.5-kb fragment containing the 5' end of the .beta.lactamase gene
and the Tacll-dsbC element was purified. This was ligated with a
4.6-kb fragment of EcoRI-digested and filled-in pBKIGF-2B, which
had also been digested with ScaI. This fragment of pBKIGF-2B
contained the 3' end of the .beta.-lactamase, allowing selection
for resistance to ampicillin as well as the DNA sequences for
resistance to tetracycline and production of IGF-I. This plasmid is
denoted pJJ40 (see FIG. 2).
This plasmid pJJ40 was transformed into E. coli strain 33D3 ,
described above. 33D3 cells containing pJJ40 were then grown in a
10-liter fermentor under conditions identical to IGF-I production
as described in U.S. Pat. No. 5,304,472, Example II, 1.times.feed
conditions. When the optical density (A.sub.550) of the culture
reached 60, 1% lactose was added to induce the tacII promoter,
thereby increasing dsbC expression. The growth medium was designed
for phosphate depletion and phoA promoter induction to occur at an
optical density (A.sub.550) of 80 to 100. Total IGF-I production
was measured by an HPLC assay described by U.S. Pat. No.
5,304,472.
The results are that the 1% lactose produced 4 g/L IGF-I, versus
the control, pBKIGF-2B, no inducer, shown in Table 1 above, which
produced 3 g/L of IGF-I. The correctly folded IGF-I can be
recovered in high yield by liquid-liquid extraction followed by
aerobic refolding as described above.
EXAMPLE III
The dsbA gene was cloned into a vector pBAD18 (FIG. 3) containing a
cloned inducible arabinose promoter (Guzman et al., supra) that
over-expresses the gene in response to the presence of arabinose in
the medium. The dsbA gene was amplified by PCR, which attached
restriction sites at the ends for ease in cloning. An EcoRI site
was placed at the 5' end and a KpnI site at the 3' end. The vector
into which dsbA was cloned was digested with the same enzymes and
the dsbA PCR product was ligated with the digested vector to form
pBADJ2 (FIG. 3). This plasmid pBADJ2 was digested with ClaI and
HindIII and then made blunt-ended with the use of Klenow polymerase
fragment I. A 1.9-kb fragment encoding the arabinose promoter and
dsbA was purified. This arabinose promoter with the dsbA gene was
then cloned into the EcoRV site of the IGF-I production plasmid
pBKIGF-2B as described above. The direction of transcription of
dsbA was the same as for IGF-I. This plasmid was designated as
pBKIGF2B-A.
The plasmid pBKIGF2B-A was then transformed into E. coli strain
36F8 (with the genotype W3110 tonA phoA.DELTA. (argF-lac)ptr3 degP
kan.sup.R ilvG30 , and being temperature resistant at 37.degree.
C.). This strain was constructed in several steps using the same
basic techniques described above. The starting host for the
construction, 27A7, is described in detail in U.S. Pat. No.
5,304,472.
Next, the degP41 kan.sup.r mutation was introduced into 27A7. This
mutation is described in U.S. Pat. No. 5,304,472. The resulting
strain is designated 27B4. The 27B4 strain grows poorly on LB media
at 37.degree. C. and 42.degree. C. A temperature-resistant isolate
was obtained at 37.degree. C. by picking a spontaneous colony that
grew better. This strain is designated 35E7.
A ribose deletion was then introduced into strain 35E7 by P1
co-transduction with a linked Tn10 insertion in the ilv gene. The
isoleucine/valine auxotrophy was transduced to prototrophy using P1
phage grown on a strain carrying the ilvG2096.sup.R mutation
(Lawther et.al., Proc. Natl. Acad. Sci. USA, 78:922-925 [1981]),
which repairs a frameshift that causes the wild-type E. coli K-12
strain to be sensitive to valine. The ilv2096.sup.R locus was
confirmed by resistance of the 36F8 host to 40 .mu.g/mL valine. The
rbs deletion was also repaired during this step, resulting in a
strain able to utilize ribose as a carbon source.
The final strain 36F8 is resistant to T1 and .O slashed.80 phage,
lacks two proteases, does not overproduce alkaline phosphatase upon
depletion of phosphate in the medium, grows well at 37.degree. C.,
and is not susceptible to valine toxicity.
36F8 cells containing the plasmid pBKIGF2B-A were then grown in a
10-liter fermentor under conditions identical to the IGF-I
production described in U.S. Pat. No. 5,304,472, supra,
1.times.feed conditions, Example III. When the optical density
(A.sub.550) of the culture reached 80, 1% (w/v) L-arabinose was
added. This induced dsbA expression and resulted in secretion of
the DsbA protein into the periplasm. The growth medium was designed
for phosphate depletion and phoA promoter induction to occur at an
optical density (A.sub.550) of 100. Total IGF-I production was
measured by the HPLC assay used in Example I.
The results are shown in Table 2 below:
TABLE 2 ______________________________________ Fermentation Run
Conditions g/L IGF-I ______________________________________ 1
pBKIGF2B-A, 1.5% arabinose 6.1 2 pBKIGF2B-A, 1.0% arabinose 7.6 3
pBKIGF2B-A, 1.0% arabinose 7.2 4 pBKIGF2B-A, 0.5% arabinose 6.6 5
typical periplasmic IGF-I 3.7 process
______________________________________
The results show that over-expression of the dsbA gene in the
periplasm of the bacteria improves yield of total (soluble and
insoluble) IGF-I for all rims involving the arabinose promoter and
DsbA. This increase in IGF-I accumulation was approximately 2-fold
using 1% L-arabinose. The IGF-I in correctly folded form can be
recovered by liquid-liquid extraction and by refolding as described
above, or by methods as described in U.S. Pat. No. 5,288,931.
The yield increase was not seen for DsbB, as cells over-expressing
dsbB from a very similar plasmid do not show the increase.
Furthermore, if the dsbA DNA and IGF-I DNA were co-expressed using
the same promoter, total IGF-I yields were decreased as compared to
a control with no dsbA gene (pRKIGF-2B).
* * * * *